The concept of apoptosis was first proposed in 1972 by Kerr et al, and refers to a well organized and autonomous death form different from cell necrosis, which is controlled by multiple genes. It plays an indispensable role in the evolution, homeostatic process and development of multiple systems of organisms (Renehan et al., 2001; Nijhawan et al., 2000; Opferman and Korsmeyer, 2003; Osborne, 1996). The mechanism of apoptosis is highly complicated, and involves the implication of a series of energy-dependent cascade reactions. Till now, two major apoptotic pathways have been found: extrinsic pathway or death receptor pathway and intrinsic pathway or mitochondrial pathway, and there is a link between the two pathways (Igney and Krammer, 2002); there is another pathway known as perforin/granzyme pathway, whereby granzyme A or granzyme B is involved in T cell-mediated cytotoxicity and perforin-granzyme dependent cell killing. Finally, the extrinsic pathway, intrinsic pathway and granzyme B pathway converge to the same execution pathway, i.e. caspase-3 cleavage, DNA breakage, cytoskeletal and nucleoprotein degradation, protein crosslink, formation of apoptotic bodies, expression of ligand of phagocytic cell receptor, and eventually being swallowed. The apoptosis triggered by extrinsic pathway requires the interaction between ligands and transmembrane death receptors, and these death receptors belong to the tumor necrosis factor (TNF) receptor superfamily (Locksley et al., 2001). The members of TNF receptor superfamily share similar cysteine-rich extracellular binding domain and an intracellular death domain with approximately 80 amino acids (Ashkenazi and Dixit, 1998). Death domain plays a key role in the transmission of death signals from the cell surface to inside of the cell, at present, the well-established ligand/death receptor pathways include FasL/FasR, TNF-α/TNFR1, Apo3L/DR3, Apo2L/DR4 and Apo2L/DR5 (Chicheportiche et al., 1997; Ashkenazi et al., 1998; Peter and Kramer, 1998; Suliman et al., 2001; Rubio-Moscardo et al., 2005).
Apoptosis occurs under many physiological conditions, such as embryonic development, and clonal selection in the immune system (Itoh et al., 1991). Apoptosis under the physiological conditions is controlled by precise regulation, and excessively upregulation or downregulation of apoptosis will result in pathological changes, such as developmental defects, autoimmune disease, neurodegenerative disease, or malignancy and the like. Malignancy is now considered as the result of excessive cell proliferation and/or decrease of cell removal due to the dysfunction of the normal cell cycle control mechanisms (King and Cidlowski, 1998). The inhibition of apoptosis plays a key role in the onset and development of some tumors (Kerr et al., 1994).
In tumor cells, its apoptosis can be inhibited through a variety of molecular mechanisms, such as the expression of anti-apoptotic protein, downregulation of expression of pro-apoptotic proteins or inactivation of mutation of pro-apoptotic protein. Based on the understanding of the role of apoptosis in the onset and development of tumor and signal transduction pathways of apoptosis, drugs promoting apoptosis of tumor cells have been developed or are developed. The development of the novel drugs targeting extracellular apoptotic pathway of the death receptor is one research focus of recent years, in particular, DR4/DR5. Several drug candidates targeting DR4/DR5 are now in clinical trials.
TNF related apoptosis inducing ligand (TRAIL) gene was first cloned and named by Wiley, et al. in 1995. In 1996, the same gene was cloned and named as Apo2L by Pitti et al. TRAIL/Apo2L is widely expressed in various tissues of normal human (lung, liver, kidney, spleen, thymus, prostate, ovary, small intestine, peripheral lymphocytes, heart, placenta, skeletal muscle, etc.) (Wiley et al., 1995; Pitti et al., 1996). TRAIL/Apo2L exists in vivo in two forms, i.e., membrane-bound and soluble TRAIL/Apo2L, both of which can form a stable homotrimer and bind with receptor to perform biological effects. A large number of in vivo and in vitro experiments show that, TRAIL/Apo2L can selectively induce apoptosis of several tumor cells and transformed cells; application of recombinant TRAIL/Apo2L protein in tumor-bearing animals can significantly inhibit tumor cell growth and even result in tumor regression without obvious damage to the host. The specificity, efficiency and non-toxicity of TRAIL/Apo2L killing tumor cells are significantly advantageous than that of CD95L and TNFα in the same family, since the latter can lead to the systemic and hard-to-be-controlled inflammation and severe toxicity such as degeneration, necrosis, hemorrhage to liver tissue, and even death (Tartaglia L, 1992). The anti-tumor activity and safety of TRAIL/Apo2L are significantly advantageous than the clinically used radiotherapy and chemotherapy etc. Animal experiments have confirmed that, TRAIL/Apo2L combined with radiotherapy and chemotherapy can produce a synergistic effect, thereby reducing the dosage and side effects of the latter. Therefore, TRAIL/Apo2L is now considered as the most promising anticancer drugs. Immunex Inc. (U.S.) discloses the gene sequence, expression vectors, and host cells of the wild-type TRAIL, and anti-TRAIL antibody (US6284236). Genentech, Inc. (U.S.) discloses a method of using wild-type APO2L for treatment of breast, colon, lung, prostate and glioma cancer (US6030945, US6746668, US6998116). The other two patent applications to Genentech Inc. disclose certain amino acid positions in APO2L polypeptide were replaced (US6740739; WO 03/029420A2). Due to low activity of the recombinantly prepared soluble wild type TRAIL/APO2L, it is not applicable for industrial and clinical applications, therefore, the structure of wild-type TRAIL/APO2L is reconstructed and modified to acquire a permuted TRAIL/APO2L with high activity is a main way to develop these drugs.
WO2005/042744 discloses a circularly permuted form of TRAIL, which has a significant selective inhibitory effect on tumor.